Natural killer T cells (NKT cells) play an important role in regulating immune responses. NKT cells are stimulate by glycolipid antigens presented by CD1d, and in the last few years significant advances have been made in understanding this process. Natural antigens for NKT cells have been identified, and crystal structures of CD1d bound to glycolipids have been solved. As factors leading to NKT cell stimulation are elucidated, the importance of glycolipid trafficking and specific interactions with T cell receptors (TCRs) is emerging. However, glycolipid trafficking and interactions with TCRs are not well understood. Proposed research includes development of labeled glycolipids for use in studying specific trafficking events; specifically, the influences of structural variations of glycolipids on trafficking and how differences in trafficking influence the Th1/Th2 bias of cytokine release by NKT cells. Studies of glycolipid and CD1d interactions with TCRs will involve incremental structural modifications of known antigens to determine requirements for association and stimulation. In addition, glycolipid functionality has bee identified that can be modified without impacting negatively NKT cell stimulation, and this information provides a means of using small molecules appended to glycolipids to modify the affinity of TCRs for glycolipid-CD1d complexes. It is anticipated that higher affinity will result in prolonged stimulation of NKT cells and increased cytokine release; however, the impact of this affinity on cytokine release profiles is not known and these studies will provide that information. The number of known organisms producing natural antigens for NKT cells is rather small, and bacteria related to those know to stimulate NKT cell directly will be screened for NKT stimulatory behavior. Structures of antigens will then be determined and confirmed through total synthesis. Relevance to Public Health: Responses of NKT cells influence disease states including infection, tumor rejection, and autoimmune diseases. An understanding of how glycolipids stimulate different responses from NKT cells will facilitate use of these responses to improve human health. Proposed research will increase this understanding while augmenting the arsenal of glycolipids that stimulate NKT cells.